Ligand functionalized polymers

ABSTRACT

A method of separating a target biological species from a fluid is disclosed comprising contacting the fluid with a ligand-functionalized polymer comprising:
     a water soluble or water dispersible aminopolymer functionalized with guanidinyl groups;   whereby a complex comprising the functionalized substrate and the target biological species is formed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/310,005, filed Mar. 3, 2010, the disclosure of whichis incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present disclosure relates to ligand-functionalized polymers, andmethods for preparing the same. The functionalized polymers are usefulin selectively binding and removing biological materials, such asviruses, from biological samples.

BACKGROUND

Detection, quantification, isolation and purification of targetbiomaterials, such as viruses and biomacromolecules (includingconstituents or products of living cells, for example, proteins,carbohydrates, lipids, and nucleic acids) have long been objectives ofinvestigators. Detection and quantification are importantdiagnostically, for example, as indicators of various physiologicalconditions such as diseases. Isolation and purification ofbiomacromolecules are important for therapeutic uses and in biomedicalresearch. Biomacromolecules such as enzymes which are a special class ofproteins capable of catalyzing chemical reactions are also usefulindustrially; enzymes have been isolated, purified, and then utilizedfor the production of sweeteners, antibiotics, and a variety of organiccompounds such as ethanol, acetic acid, lysine, aspartic acid, andbiologically useful products such as antibodies and steroids.

In their native state in vivo, structures and corresponding biologicalactivities of these biomacromolecules are maintained generally withinfairly narrow ranges of pH and ionic strength. Consequently, anyseparation and purification operation must take such factors intoaccount in order for the resultant, processed biomacromolecule to havepotency.

The use of certain ionic polymers, especially cationic polymers, for theflocculation of cell and/or cell debris, as well as for theprecipitation of proteins, is known. Similarly, ionic polymers have beenused to modify filtration media to enhance the removal of impuritiesfrom process streams in depth filtration or membrane absorber typeapplications. The effectiveness of these flocculants is typicallyreduced as the conductivity of the media being processed increases, i.e.as the salt content increases. There is a need in the art for polymericmaterials with increased affinity for biological species under highionic strength conditions.

Chromatographic separation and purification operations can be performedon biological product mixtures, based on the interchange of a solutebetween a moving phase, which can be a gas or liquid, and a stationaryphase. Separation of various solutes of the solution mixture isaccomplished because of varying binding interactions of each solute withthe stationary phase; stronger binding interactions generally result inlonger retention times when subjected to the dissociation ordisplacement effects of a mobile phase compared to solutes whichinteract less strongly and, in this fashion, separation and purificationcan be effected.

Most current capture or purification chromatography is done viaconventional column techniques. These techniques have severebottlenecking issues in downstream purification, as the throughput usingthis technology is low. Attempts to alleviate these issues includeincreasing the diameter of the chromatography column, but this in turncreates challenges due to difficulties of packing the columnseffectively and reproducibly. Larger column diameters also increase theoccurrence of problematic channeling. Also, in a conventionalchromatographic column, the absorption operation is shut down when abreakthrough of the desired product above a specific level is detected.This causes the dynamic or effective capacity of the adsorption media tobe significantly less than the overall or static capacity. Thisreduction in effectiveness has severe economic consequences, given thehigh cost of some chromatographic resins.

Polymeric resins are widely used for the separation and purification ofvarious target compounds. For example, polymeric resins can be used topurify or separate a target compound based on the presence of an ionicgroup, based on the size of the target compound, based on a hydrophobicinteraction, based on an affinity interaction, or based on the formationof a covalent bond. There is a need in the art for polymeric substrateshaving enhanced affinity for viruses and other biological species toallow selective removal from a biological sample. There is further needin the art for ligand functionalized membranes that overcome limitationsin diffusion and binding, and that may be operated at high throughputand at lower pressure drops.

SUMMARY OF THE INVENTION

The present invention is directed to ligand-functionalized polymers, andmethods of making the same. More specifically, the ligand-functionalizedpolymer includes a polyamine polymer, which has been modified to providegrafted ligand groups having the requisite affinity for binding neutralor negatively charged biomaterials, such as cells, cell debris,bacteria, spores, viruses, nucleic acids, and proteins.

In some embodiments, the ligand-functionalized polymer may be used as aflocculant whereby a biological sample, such as a cell culture fluid, iscontacted causing negative and/or neutral species to bind to the polymerand precipitate from the solution or suspension. In another embodiment,a base substrate, such as a microporous membrane, may be coated with theligand-functionalized polymer.

Methods of making a ligand functionalized substrate are provided. Insome embodiments, the method comprises reacting a polyamine polymer witha guanylating agent, optionally in the presence of an acid catalyst.

A functionalized polymer is provided, having grafted pendent ligandgroups, of the formula:

wherein

-   R² is a H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or a residue of the    polymer chain;-   each R³ is independently H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,-   each R⁴ is H, C₁-C₁₂ alkyl or alkylene, C₅-C₁₂ (hetero)aryl or    (hetero)arylene, cyano, or —C(═NH)—N(R²)-Polymer, and-   n is 1 or 2.

It will be recognized that the “Polymer-N(R²)—” group of Formula I isthe linkage formed between an amine group of polyamino polymer and theguanylating agent.

As used herein, “alkyl” or “alkylene” includes straight-chained,branched, and cyclic alkyl groups and includes both unsubstituted andsubstituted alkyl groups. Unless otherwise indicated, the alkyl groupstypically contain from 1 to 20 carbon atoms. Examples of “alkyl” as usedherein include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, isobutyl, t-butyl, isopropyl, n-octyl, n-heptyl,ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, andnorbornyl, and the like. Unless otherwise noted, alkyl groups may bemono- or polyvalent.

As used herein, “aryl” or “arylene” is an aromatic group containing 5-12ring atoms and can contain optional fused rings, which may be saturated,unsaturated, or aromatic. Examples of an aryl groups include phenyl,naphthyl, biphenyl, phenanthryl, and anthracyl. Heteroaryl is arylcontaining 1-3 heteroatoms such as nitrogen, oxygen, or sulfur and cancontain fused rings. Some examples of heteroaryl groups are pyridyl,furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, and benzthiazolyl. Unless otherwise noted, aryl andheteroaryl groups may be mono- or polyvalent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 are plots of the Geobacillus stearothermophilusflocculation data of Example 35.

DETAILED DESCRIPTION OF THE INVENTION

In the article and methods of this invention, ligand-functionalizedpolymers are provided which have enhanced affinity, especially in highionic strength media, for neutral or negatively charged biologicalmaterials such as host cell proteins, DNA, RNA, viruses, and othermicroorganisms. The affinity for such biomaterials allows positivelycharged materials, such as antibodies, to be purified, as they are notbound to the ligand functional groups. The ligand functionalizedsubstrate allows the selective capture or binding of target biomaterialsby the ligand groups, while other materials, lacking the affinity forthe ligand groups are passed. In some embodiments the ligandfunctionalized polymer is used as a flocculant to selectively bindtarget biomaterials, precipitate them from solution, and theprecipitated adduct subsequently separated.

Polyamine Polymer

The base polymer comprises a polyamine polymer; i.e. a polymer havingprimary or secondary amino groups that may be pendent or catenary, i.e.in the polymer chain. The aminopolymers contain primary or secondaryamine groups and can be prepared by chain growth or step growthpolymerization procedures with the corresponding monomers. Thesemonomers can also, if desired, be copolymerized with other monomers. Thepolymer can also be a synthesized or naturally occurring biopolymer. Ifany of these polymers, irrespective of source, do not contain primary orsecondary amine groups, these functional groups can be added by theappropriate graft chemistry.

Useful aminopolymers are water soluble or water-dispersible. As usedherein, the term “water soluble” refers to a material that can bedissolved in water. The solubility is typically at least about 0.1 gramper milliliter of water. As used herein, the term “water dispersible”refers to a material that is not water soluble but that can beemulsified or suspended in water.

Examples of amino polymers suitable for use, which are prepared by chaingrowth polymerization include, but are not limited to: polyvinylamine,poly(N-methylvinylamine), polyallylamine, polyallylmethylamine,polydiallylamine, poly(4-aminomethylstyrene), poly(4-aminostyrene),poly(acrylamide-co-methylaminopropylacrylamide), andpoly(acrylamide-co-aminoethylmethacrylate).

Examples of amino polymers suitable for use, which are prepared by stepgrowth polymerization include, but are not limited to: polyethylenimine,polypropylenimine, polylysine, polyaminoamides,polydimethylamine-epichlorohydrin-ethylenediamine, and any of a numberof polyaminosiloxanes, which can be built from monomers such asaminopropyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,N-trimethoxysilylpropyl-N-methylamine, andbis(trimethoxysilylpropyl)amine.

Useful aminopolymers that have primary or secondary amino end groupsinclude, but are not limited to, those formed from polyamidoamine(PAMAM) and polypropylenimine: e.g. DAB-Am and PAMAM dendrimers (orhyperbranched polymers containing the amine or quaternary nitrogenfunctional group). Exemplary dendrimeric materials formed from PAMAM arecommercially available under the trade designation Starburst™ (PAMAM)dendrimer” (e.g., Generation 0 with 4 primary amino groups, Generation 1with 8 primary amino groups, Generation 2 with 16 primary amino groups,Generation 3 with 32 primary amino groups, and Generation 4 with 64primary amino groups) from Aldrich Chemical, Milwaukee, Wis. Dendrimericmaterials formed from polypropylenimine is commercially available underthe trade designation “DAB-AM” from Aldrich Chemical. For example,DAB-Am-4 is a generation 1 polypropylenimine tetraamine dendrimer with 4primary amino groups, DAB-Am-8 is a generation 2 polypropylenimineoctaamine dendrimer with 8 primary amino groups, DAB-Am-16 is ageneration 3 polypropylenimine hexadecaamine with 16 primary aminogroups, DAB-Am-32 is a generation 4 polypropylenimine dotriacontaaminedendrimer with 32 primary amino groups, and DAB-Am-64 is a generation 5polypropylenimine tetrahexacontaamine dendrimer with 64 primary aminogroups.

Examples of aminopolymers suitable for use, which are biopolymersinclude chitosan, and starch, where the latter is grafted with reagentssuch as methylaminoethylchloride.

Other categories of aminopolymers suitable for use includepolyacrylamide homo- or copolymers with amino monomers includingaminoalkyl(meth)acrylate, (meth)acrylamidoalkylamine, and diallylamine

Preferred aminopolymers include polyaminoamides, polyethyleneimine,polyvinylamine, polyallylamine, and polydiallylamine

Suitable commercially available aminopolymers include, but are notlimited to, polyamidoamines such as ANQUAMINE™ 360, 401, 419, 456, and701 (Air Products and Chemicals, Allentown, Pa.); LUPASOL™polyethylenimine polymers such as FG, PR 8515, Waterfree, P, PS (BASFCorporation, Resselaer, N.Y.); polyethylenimine polymers such as CORCAT™P-600 (EIT Company, Lake Wylie, S.C.); polyoxyalkyleneamines such asJEFFAMINE.™ D-230, D-400, D-2000, HK-511 (XTJ-511), XTJ-510 (D-4000),XTJ-500 (ED-600), XTJ-502 (ED-2003), T-403, XTJ-509 (T-3000), and T-5000(Huntsman Corporation, Houston, Tex.); and polyamide resins such as theVERSAMID series of resins that are formed by reacting a dimerizedunsaturated fatty acid with alkylene diamines (Cognis Corporation,Cincinnati, Ohio).

The ligand functional polymer may be prepared by condensation of thepolyamine polymer with a guanylating agent. Known guanylating agentsinclude: cyanamide; O-alkylisourea salts such as O-methylisoureasulfate, O-methylisourea hydrogen sulfate, O-methylisourea acetate,O-ethylisourea hydrogen sulfate, and O-ethylisourea hydrochloride;chloroformamidine hydrochloride; 1-amidino-1,2,4-triazole hydrochloride;3,5-dimethylpyrazole-1-carboxamidine nitrate; pyrazole-1-carboxamidinehydrochloride; N-amidinopyrazole-1-carboxamidine hydrochloride; andcarbodiimides, such as dicyclohexylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, anddiisopropylcarbodiimide. The polyamine polymer may also be acylated withguanidino-functional carboxylic acids such as guanidinoacetic acid and4-guanidinobutyric acid in the presence of activating agents such as EDC(N[-3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride), or EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline). Additionally, theligand functional polymer may be prepared by alkylation withchloroacetone guanyl hydrazone, as described in U.S. Pat. No. 5,712,027.

Reagents for the preparation of biguanide-functional polymers includesodium dicyanamide, dicyanodiamide and substituted cyanoguanidines suchas N³-p-chlorophenyl-N¹-cyanoguanidine, N³-phenyl-N¹-cyanoguanidine,N³-alpha-naphthyl-N¹-cyanoguanidine, N³-methyl-N¹-cyanoguanidine,N³,N³-dimethyl-N¹-cyanoguanidine, N³-(2-hydroxyethyl)-N¹-cyanoguanidine,and N³-butyl-N¹-cyanoguanidine. Alkylene- and arylenebiscyanoguanidinesmay be utilized to prepare biguanide functional polymers by chainextension reactions. The preparation of cyanoguanidines andbiscyanoguanidines is described in detail in Rose, F. L. and Swain, G.J. Chem Soc., 1956, pp. 4422-4425. Other useful guanylating reagents aredescribed by Alan R. Katritzky et al., Comprehensive Organic FunctionalGroup Transformation, Vol. 6, p. 640. Generally, such guanylationreagents are used in amounts sufficient to functionalize 0.5 to 100 molepercent, preferably 2.5 to 50 mole percent, of the available aminogroups of the aminopolymer.

The resulting polymer will have pendent or catenary guanidinyl groups ofthe formula:

wherein

-   R² is a H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or a residue of the    polymer chain;-   each R³ is independently H, C₁-C₁₂ alkyl, or C₅-C₁₂ (hetero)aryl,-   each R⁴ is H, C₁-C₁₂ alkyl or alkylene, C₅-C₁₂ (hetero)aryl or    (hetero)arylene, cyano, or —C(═NH)—N(R²)-Polymer, and-   n is 1 or 2.

In some embodiments, it may be advantageous to functionalize the aminecontaining polymer with other ligands in addition to the guanidinylligand. For example, it may be useful to include a hydrophobic ligand,an ionic ligand, or a hydrogen bonding ligand. This can be particularlyadvantageous for the capture of certain biological species, especiallyunder conditions of high ionic strength.

The additional ligands are readily incorporated into the ligandfunctional polymers by alkylation or acylation procedures well known inthe art, such as by using halide, sulfonate, or sulfate displacementreactions, or by using epoxide ring opening reactions. Useful alkylatingagents for these reactions include, for example, dimethylsulfate, butylbromide, butyl chloride, benzyl bromide, dodecyl bromide,2-chloroethanol, bromoacetic acid, 2-chloroethyltrimethylammoniumchloride, styrene oxide, glycidyl hexadecyl ether,glycidyltrimethylammonium chloride, and glycidyl phenyl ether. Usefulacylating agents include, for example, acid chlorides and anhydridessuch as benzoyl chloride, acetic anhydride, succinic anhydride, anddecanoyl chloride, and isocyanates such as trimethylsilylisocyanate,phenyl isocyanate, butyl isocyanate, and butyl isothiocyanate. In suchembodiments 0.1 to 20 mole percent, preferably 2 to 10 mole percent, ofthe available amino groups of the aminopolymer may be alkylated and/oracylated.

The disclosure further provides a functionalized substrate comprising abase substrate and an ungrafted coating of the ligand functionalizedpolymer thereon. Preferably the base substrate is a porous basesubstrate having interstitial and outer surfaces.

The base substrate may be formed from any suitable metallic,thermoplastic, or thermoset material. The material may be an organic orinorganic polymeric material. Suitable organic polymeric materialsinclude, but are not limited to, poly(meth)acrylates,poly(meth)acrylamides, polyolefins, poly(isoprenes), poly(butadienes),fluorinated polymers, chlorinated polymers, polyamides, polyimides,polyethers, poly(ether sulfones), poly(sulfones), polyvinyl acetates),copolymers of vinyl acetate, such as poly(ethylene)-co-polyvinylalcohol), poly(phosphazenes), polyvinyl esters), polyvinyl ethers),polyvinyl alcohols), and poly(carbonates). Suitable inorganic polymericmaterials include, but are not limited to, quartz, silica, glass,diatomaceous earth, and ceramic materials.

Suitable polyolefins include, but are not limited to, poly(ethylene),poly(propylene), poly(1-butene), copolymers of ethylene and propylene,alpha olefin copolymers (such as copolymers of ethylene or propylenewith 1-butene, 1-hexene, 1-octene, and 1-decene),poly(ethylene-co-1-butene) and poly(ethylene-co-1-butene-co-1-hexene).

Suitable fluorinated polymers include, but are not limited to,poly(vinyl fluoride), poly(vinylidene fluoride), copolymers ofvinylidene fluoride (such as poly(vinylidenefluoride-co-hexafluoropropylene), and copolymers ofchlorotrifluoroethylene (such aspoly(ethylene-co-chlorotrifluoroethylene).

Suitable polyamides include, but are not limited to,poly(iminoadipoyliminohexamethylene),poly(iminoadipoyliminodecamethylene), and polycaprolactam. Suitablepolyimides include, but are not limited to, poly(pyromellitimide).

Suitable poly(ether sulfones) include, but are not limited to,poly(diphenylether sulfone) and poly(diphenylsulfone-co-diphenyleneoxide sulfone).

Suitable copolymers of vinyl acetate include, but are not limited to,poly(ethylene-co-vinyl acetate) and such copolymers in which at leastsome of the acetate groups have been hydrolyzed to afford variouspoly(vinyl alcohols).

The base substrate may be in any form such as particles, fibers, filmsor sheets. Suitable particles include, but are not limited to, magneticparticles, organic particles, inorganic particles, and porous andnonporous particles. Preferably the base substrate is porous. Suitableporous base substrates include, but are not limited to, porousparticles, porous membranes, porous nonwoven webs, and porous fibers.

In some embodiments, the porous base substrate is formed from propylenehomo- or copolymers, most preferably propylene homopolymers.Polypropylene polymers are often a material of choice for porousarticles, such as nonwovens and microporous films, due to propertiessuch as non-toxicity, inertness, low cost, and the ease with which itcan be extruded, molded, and formed into articles.

In many embodiments, the porous base substrate has an average pore sizethat is typically greater than about 0.2 micrometers in order tominimize size exclusion separations, minimize diffusion constraints andmaximize surface area and separation based on binding of a targetmolecule. Generally, the pore size is in the range of 0.1 to 10micrometers, preferably 0.5 to 3 micrometers and most preferably 0.8 to2 micrometers when used for binding of viruses. The efficiency ofbinding other target molecules may confer different optimal ranges.

Suitable porous base substrates include, but are not limited to, porousand microporous membranes, nonwoven webs, and fibers. In someembodiments, the porous base substrate is a microporous membrane such asa thermally-induced phase separation (TIPS) membrane. TIPS membranes areoften prepared by forming a homogenous solution of a thermoplasticmaterial and a second material above the melting point of thethermoplastic material. Upon cooling, the thermoplastic materialcrystallizes and phase separates from the second material. Thecrystallized thermoplastic material is often stretched. The secondmaterial is optionally removed either before or after stretching.Microporous membrane are further disclosed in U.S. Pat. No. 4,539,256(Shipman), U.S. Pat. No. 4,726,989 (Mrozinski), U.S. Pat. No. 4,867,881(Kinzer), U.S. Pat. No. 5,120,594 (Mrozinski), U.S. Pat. No. 5,260,360(Mrozinski et al.), and U.S. Pat. No. 5,962,544 (Waller), all of whichare assigned to 3M Company (St. Paul, Minn.). Further, the microporousfilm can be prepared from ethylene-vinyl alcohol copolymers as describedin U.S. Pat. No. 5,962,544 (Waller).

Some exemplary TIPS membranes comprise poly(vinylidene fluoride) (PVDF),polyolefins such as polyethylene homo- or copolymers or polypropylenehomo- or copolymers, vinyl-containing polymers or copolymers such asethylene-vinyl alcohol copolymers and butadiene-containing polymers orcopolymers, and acrylate-containing polymers or copolymers. For someapplications, a TIPS membrane comprising PVDF is particularly desirable.TIPS membranes comprising PVDF are further described in U.S. Pat. No.7,338,692 (Smith et al.).

In another exemplary embodiment the porous bases substrate comprises anylon microporous film or sheet, such as those described in U.S. Pat.No. 6,056,529 (Meyering et al.), U.S. Pat. No. 6,267,916 (Meyering etal.), U.S. Pat. No. 6,413,070 (Meyering et al.), U.S. Pat. No. 6,776,940(Meyering et al.), U.S. Pat. No. 3,876,738 (Marinacchio et al.), U.S.Pat. Nos. 3,928,517, 4,707,265 (Knight et al.), and U.S. Pat. No.5,458,782 (Hou et al.).

In other embodiments, the porous base substrate is a nonwoven web whichmay include nonwoven webs manufactured by any of the commonly knownprocesses for producing nonwoven webs. As used herein, the term“nonwoven web” refers to a fabric that has a structure of individualfibers or filaments which are randomly and/or unidirectionally interlaidin a mat-like fashion.

For example, the fibrous nonwoven web can be made by wet laid, carded,air laid, spunlaced, spunbonding or melt-blowing techniques orcombinations thereof. Spunbonded fibers are typically small diameterfibers that are formed by extruding molten thermoplastic polymer asfilaments from a plurality of fine, usually circular capillaries of aspinneret with the diameter of the extruded fibers being rapidlyreduced. Meltblown fibers are typically formed by extruding the moltenthermoplastic material through a plurality of fine, usually circular,die capillaries as molten threads or filaments into a high velocity,usually heated gas (e.g. air) stream which attenuates the filaments ofmolten thermoplastic material to reduce their diameter. Thereafter, themeltblown fibers are carried by the high velocity gas stream and aredeposited on a collecting surface to from a web of randomly disbursedmeltblown fibers. Any of the non-woven webs may be made from a singletype of fiber or two or more fibers that differ in the type ofthermoplastic polymer and/or thickness.

Further details on the manufacturing method of non-woven webs of thisinvention may be found in Wente, Superfine Thermoplastic Fibers, 48INDUS. ENG. CHEM. 1342(1956), or in Wente et al., Manufacture OfSuperfine Organic Fibers, (Naval Research Laboratories Report No. 4364,1954).

In one embodiment the base substrate may have a coating of the ligandfunctional (co)polymer on a surface thereon. Useful coating techniquesinclude applying a solution or dispersion of the (co)polymer, optionallyincluding a crosslinker, onto the base substrate. Polymer application isgenerally followed by evaporating the solvent to form the polymercoating. Coating methods include the techniques commonly known as dip,spray, knife, bar, slot, slide, die, roll, or gravure coating. Coatingquality generally depends on mixture uniformity, the quality of thedeposited liquid layer, and the process used to dry or cure the liquidlayer.

In some embodiments, the polyamine polymer is first coated on the basesubstrate and subsequently reacted with a guanylating agent, such aspyrazole carboxamidine hydrochloride.

In other embodiments, the ligand functional (co)polymer itself is coatedon the base substrate. Useful crosslinkers in these instances includeamine reactive compounds such as bis- and polyaldehydes such asglutaraldehyde, bis- and polyepoxides such as butanedioldiglycidyletherand ethyleneglycoldiglycidylether, polycarboxylic acids and theirderivatives (e.g., acid chlorides), polyisocyanates, formaldehyde-basedcrosslinkers such as hydroxymethyl and alkoxymethyl functionalcrosslinkers, such as those derived from urea or melamine, andamine-reactive silanes, such as 3-glycidoxypropyltrimethoxysilane,3-glycidoxypropyltriethoxysilane, 5,6-epoxyhexyltriethoxysilane,(p-chloromethyl)phenyltrimethoxysilane, chloromethyltriethoxysilane,3-isocyanatopropyltriethoxysilane, and3-thiocyanatopropyltriethoxysilane.

In other embodiments, the ligand functional copolymer is coated on thebase substrate by polyelectrolyte layer-by-layer coating techniques,such as those described in EP 472,990.

In some embodiments, the base substrate has amine-reactive functionalgroups, such as halide, epoxy, ester, isocyanate groups, on the surfacethereof. These surface functional groups may react with extant aminefunctional groups on the ligand functional aminopolymer. In anotherembodiment, the surface of the base substrate may be provided withamine-reactive functional groups, that can react with the amine groupsof the ligand functionalized polymer.

The amine-reactive functional groups may be provided by any of thetechniques known to one in the art. In one embodiment the base substratemay have a coating of a (co)polymer comprising amine-reactive functionalgroups on a surface thereon. Especially useful (co)polymers in thisregard are azlactone functional (co)polymers such as those described inU.S. Pat. No. 7,101,621. Useful coating techniques include applying asolution or dispersion of the (co)polymer, optionally including acrosslinker, onto the base substrate. Polymer application is generallyfollowed by evaporating the solvent to form the polymer coating. Coatingmethods include the techniques commonly known as dip, spray, knife, bar,slot, slide, die, roll, or gravure coating. Coating quality generallydepends on mixture uniformity, the quality of the deposited liquidlayer, and the process used to dry or cure the liquid layer.

In some embodiments the polymer comprising amine-reactive groups may begrafted to the surface of a substrate by ionizing radiation-initiatedgraft polymerization of a monomer having a free-radically polymerizablegroup and a second functional group reactive with the ligand functionalpolymer, as described in Assignee's copending application (64729US002).Such monomers may include azlactone-functional monomers,isocyanatoethyl(meth)acrylate or glycidyl(meth)acrylate. Alternatively,a carbonyl functional monomer may be grafted to the surface of asubstrate by ionizing radiation-initiated graft polymerization, followedby functionalization by reaction with the ligand functional polymer ofFormula I, as described in Assignee's copending U.S. Application No.61/305,740.

The method of grafting (or coating) a ligand functionalized polymer tothe surface of the substrate alters the original nature of the basesubstrate, as the substrate bears a grafted or ungrafted coating of theligand functional polymer. The present invention enables the formationof ligand functionalized polymer substrates having many of theadvantages of a base substrate (e.g., mechanical and thermal stability,porosity), but with enhanced affinity for biological species such asviruses, resulting from the monomers and steps used to form a givenfunctionalized substrate.

The porous substrates having a coating of ligand-functionalized polymerare particularly suited as filter media, for the selective binding andremoval of target biological species including proteins, cells, celldebris, microbes, nucleic acids, and/or viruses from biological samples.The present disclosure further provides a method for the removal oftarget biological species from a biological sample by contacting thesample with the ligand polymer functionalized substrate as describedherein. As used herein “target biological species” may include acontaminant or a species of interest.

The ligand functionalized (co)polymer (either the polymer per se, or asubstrate having a coating thereof) is useful for the purification ofbiological or other fluid samples comprising biologically derivedspecies (biological species). Biological species include, but are notlimited to, cells, cell debris, proteins, nucleic acids, endotoxins, andviruses. Cells and cell debris include those derived from archaea,bacteria, and eucaryotes. Bacteria include, but are not limited to,Gram-negatives such as Pseudomonas species, Escherichia coli,Helicobacter pylori, and Serratia marcesens; Gram-positives such asStaphylococcus species, Enterococcus species, Clostridium species,Bacillus species, and Lactobacillus species; bacteria that do not staintraditionally by Gram's method such as Mycobacterium species, andnon-vegetative forms of bacteria such as spores. Eucaryotes include, butare not limited to, animal cells, algae, hybridoma cells, stem cells,cancer cells, plant cells, fungal hyphae, fungal spores, yeast cells,parasites, parasitic oocysts, insect cells, and helminthes. Proteins,include, but are not limited to, natural proteins, recombinant proteins,enzymes, and host cell proteins. Viruses include, but are not limitedto, enveloped species such as Herpesviruses, Poxviruses, Adenoviruses,Papovaviruses, Coronaviruses, retroviruses such as HIV, andPlasmaviridae; and non-enveloped species such as Caliciviridae,Corticoviridae, Myoviridae, and Picornaviridae.

In some embodiments, the biological species being removed from the fluidis the object of the purification. For example, a recombinant protein orenzyme may be prepared in cell culture or by fermentation, the(co)polymer can be added to flocculate the protein or enzyme, and theprecipitate can be separated as the first step in the purificationprocess for the protein or enzyme. In another example, the (co)polymeror a substrate with a coating thereof, may be used to capturemicroorganisms from a fluid as the first step in a process ofconcentrating, enumerating, and/or identifying the microorganisms.

In other embodiments, the biological species being removed from thefluid is a contaminant that must be removed prior to additionalprocessing steps for the fluid. The polymer can be used as a flocculantto facilitate the removal of cells and cell debris from a cell cultureor fermentation broth prior to, subsequent to, or in place of acentrifuge or depth filtration operation. For example, the (co)polymercan be used to flocculate cells in a cell culture broth prior tocentrifugation, and thereby improve the efficiency with which thecentrifugation process separates the cell mass from the liquid centrate.Alternatively, it can be added to the liquid centrate after acentrifugation step to flocculate suspended cell debris and dissolvedhost cell proteins and nucleic acids, thereby increasing the efficiencyof a subsequent depth filtration step. It can be used to flocculate orprecipitate suspended bacteria, viruses, or other microorganisms. It canbe used to precipitate either desired or contaminating proteins ornucleic acids from solution. Significantly, the ligand functional(co)polymers, or substrates having a coating thereof, are useful underconditions of high salt concentration or high ionic strength, i.e., theyare “salt tolerant”. The term “salt” is meant to include all lowmolecular weight ionic species which contribute to the conductivity ofthe solution. The importance of utility of the ligand functional(co)polymers in the presence of salt is that many process solutions usedin biopharmaceutical or enzyme manufacture have conductivities in therange of 15-30 mS/cm (approximately 150-300 mM salt) or more. Salttolerance can be measured in comparison to that of the conventionalquaternary amine or Q ligand (e.g. trimethylammonium ligand), whoseprimarily electrostatic interactions with many biological speciesrapidly deteriorates at conductivities three- to six-fold less than thetarget range. For example, membranes derivatized with the conventional Qligand exhibit a drop in φX174 viral clearance from a six log-reductionvalue (LRV) to a one (1) LRV in going from 0 to 50 mM NaCl (ca. 5-6mS/cm conductivity). Viruses such as φX174 which have pIs close to 7(are neutral or near-neutral) are extremely difficult to remove fromprocess streams. Similar problems are observed when attempting to removeother biological species from process fluids. For example, whenattempting to remove positively charged proteins such as host cellproteins through the use of filtration devices functionalized withconventional Q ligands, the process fluid may have to be dilutedtwo-fold or more in order to reduce the conductivity to an acceptablerange. This is expensive and dramatically increases the overallprocessing time.

When used as a flocculant, the amount of ligand functional polymer thatis added relative to the amount of sample can vary over a wide range.Generally, the amount added will produce a final concentration of(co)polymer in the mixture of from about 0.01 micrograms/mL to about5000 micrograms/mL. The optimal amount of polymer added will depend uponthe concentration of the species one desires to flocculate. Typically,the amount of polymer relative to the amount of species beingflocculated will be in the range of 0.01% to 100% by weight, preferably0.05%-30% by weight, more preferably about 0.1%-10% by weight. Theoptimal amount is readily assessed by challenging the sample with aseries of polymer concentrations as is well known in the art. While theabove concentration ranges are typical, one skilled in the art willrealize that other ranges may work in some instances. Flocculationefficiency also depends upon the physical and chemical characteristicsof the species being flocculated. For example, we have found thatoptimal flocculation of the near neutral virus φX174 from aqueoussuspension occurs at a polymer to virus weight ratio of about 800-1000%.

The biological sample is contacted with the ligand functionalizedpolymer (either the polymer per se, or a substrate having a coatingthereof) for a time sufficient to interact and form a complex with thetarget biological species disposed (dissolved or suspended) in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 50% of its originalconcentration. It is more preferred that the solution is contacted withthe ligand functionalized polymer for a time sufficient to interact andform a complex with the target biological species disposed in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 10% of its originalconcentration. It is still more preferred that the solution is contactedwith the ligand functionalized polymer for a time sufficient to interactand form a complex with the target biological species disposed in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 1% of its originalconcentration.

In many embodiments the ligand functionalized polymer, being positivelycharged in aqueous media, will bind near neutral or negatively chargedspecies to the ligand functional group of Formula II while other species(e.g., positively charged proteins such as monoclonal antibodies) willbe excluded or repelled from the ligand functionalized substrate. Inaddition, as previously described, the substrate may be directly orindirectly grafted with one or more ionic monomers. In particular, theligand functionalized polymer may comprise grafted ionic groups that arepositively charged at the selected pH of the biological sample solutionto enhance electrostatic charge repulsion of proteins, such asmonoclonal antibodies, many of which are charged positive at neutral pH,and ligand functional groups of Formula II to provide salt tolerance.

In some embodiments the ligand functionalized polymer and coatedsubstrate containing the bound biological species are disposable. Insuch embodiments, the binding of the biological species to the ligandfunctionalized polymer is preferably essentially irreversible becausethere is no need to recover the bound biological species. Nonetheless,if desired, one can reverse the binding of biological species byincreasing the ionic strength or changing the pH of an eluting solution.

The substrate, having a grafted or ungrafted coating of the ligandfunctionalized polymer may be any previously described, but ispreferably a microporous membrane. The membrane pore size desired isfrom 0.1 to 10 μm, preferably 0.5 to 3 micrometers and most preferably0.8 to 2 micrometers. A membrane with a high surface area for theinternal pore structure is desired, which typically corresponds to finepore sizes. However, if the pore size is too small, then the membranetends to plug with fine particulates present in the sample solution.

If desired, efficiency of binding and capture may be improved by using aplurality of stacked, ligand functionalized polymer coated porousmembranes as a filter element. Thus the present disclosure provides afilter element comprising one or more layers of the porous, ligandfunctionalized polymer coated substrate. The individual layers may bethe same or different, and may have layers of different porosity, anddegree of grafting by the aforementioned grafting monomers. The filterelement may further comprise an upstream prefilter layer and downstreamsupport layer. The individual filter elements may be planar or pleatedas desired.

Examples of suitable prefilter and support layer materials include anysuitable porous membranes of polypropylene, polyester, polyamide,resin-bonded or binder-free fibers (e.g., glass fibers), and othersynthetics (woven and non-woven fleece structures); sintered materialssuch as polyolefins, metals, and ceramics; yarns; special filter papers(e.g., mixtures of fibers, cellulose, polyolefins, and binders); polymermembranes; and others.

In another embodiment, there is provided a filter cartridge includingthe above-described filter element. In yet another embodiment, there isprovided a filter assembly comprising the filter elements and a filterhousing. In a further embodiment, this invention relates to a method ofcapture or removal of a target biological species comprising the stepsof:

a) providing the filter element comprising one of more layers of theligand functionalized base substrate of this disclosure, and

b) allowing a moving biological solution containing a target biologicalspecies to impinge upon the upstream surface of the filter element for atime sufficient to effect binding of a target biological species.

The present invention is described above and further illustrated belowby way of examples, which are not to be construed in any way as imposinglimitations upon the scope of the invention. On the contrary, it is tobe clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention and/orthe scope of the appended claims.

EXAMPLES Example 1 Alkylation of Polyethylenimine (PEI)

Dodecyl bromide (2.32 grams) was added to PEI (40 grams of a 10% byweight solution of PEI (MW=10,000, from Polysciences, Inc., Warrington,Pa.) in ethanol in an 8 ounce glass bottle and sealed. The mixture washeated in a water bath at 50° C. for 20 hours, after which time ¹H-NMRindicated complete conversion to the alkylated product

Example 2 Guanylation of Alkylated Polyetheyleneamine

A portion of the solution of alkylated product (20 grams) of Example 1was mixed with pyrazole carboxamidine hydrochloride (0.17 gram, fromSigma-Aldrich, Milwaukee, Wis.). The mixture was allowed to reactovernight at ambient temperature, after which time ¹H-NMR indicatedconversion of 2.5% of the amine groups to guanidine groups had occurred.

Examples 3-21

Similar procedures were used to produce alkylated and guanylated PEI'sas listed in Table 1.

TABLE 1 Modified Polyethylenimines PEI % % Example MW Alkylating agentAlkylated Guanylated 1 10,000 dodecyl bromide 10 0 2 10,000 dodecylbromide 10 2.5 3 10,000 dodecyl bromide 5 0 4 10,000 dodecyl bromide 52.5 5 10,000 none 0 2.5 6 10,000 none 0 12.5 7 10,000 none 0 25 8 10,000benzyl bromide 10 0 9 10,000 dimethylsulfate 10 0 10 10,000 butylbromide 10 0 11 70,000 none 0 10 12 70,000 none 0 25 13 70,000 none 0 5014 70,000 butyl bromide 10 0 15 70,000 dodecyl bromide 10 0 16 70,000benzyl bromide 10 0 17 70,000 dimethylsulfate 10 0 18 70,000 butylbromide 10 50 19 70,000 dodecyl bromide 10 50 20 70,000 benzyl bromide10 50 21 70,000 dimethylsulfate 10 50

Example 22-32 Modified Poly(Allylamine)s

Using procedures similar to those described in Example 1,poly(allylamine) (MW 60,000, Polysciences) was reacted with a variety ofalkylating agents and pyrazole carboxamidine hydrochloride to provide aseries of alkylated, guanylated, or alkylated and guanylated polymers(Table 2).

TABLE 2 Modified Poly(allylamines) % % Example Alkylating agentAlkylated Guanylated 22 dodecyl bromide 10 0 23 butyl bromide 10 0 24benzyl bromide 10 0 25 dimethylsulfate 10 0 26 none 0 10 27 none 0 25 28none 0 50 29 dodecyl bromide 10 50 30 butyl bromide 10 50 31 benzylbromide 10 50 32 dimethylsulfate 10 50

Comparative Example 1 Poly(MethacrylamidopropyltrimethylammoniumChloride) (pMAPTAC)

MAPTAC (160 grams of a 50% by weight solution in water, from Aldrich,Milwaukee, Wis.), ethanol (40 grams) and sodium persulfate (0.4 gram)were charged to a 16 ounce glass bottle. The mixture was purged with aslow stream of nitrogen gas for 10 minutes, sealed, and then tumbled ina water bath equilibrated to 55° C. for 24 hours to convert the monomerto polymer. This polymer solution was diluted with deionized water (80grams) and ethanol (40 grams) and mixed well. A sample for evaluation asa flocculant was prepared by dilution of a portion of this polymer to 1%solids by weight with deionized water, pH 7.

Example 33

A solution of bovine serum albumin (BSA, Sigma-Aldrich) was prepared in10 mM MOPS, pH 7.5, and determined to have a concentration of BSA of4.02 mg/mL. A series of BSA solutions were prepared containing variousconcentrations of sodium chloride according to Table 3.

TABLE 3 BSA Solutions [NaCl] BSA 5 M MOPS (mM, solution NaCl bufferfinal) (mL) (μL) (μL) 0 10 0 500 50 10 100 400 100 10 200 300 150 10 300200 200 10 400 100 250 10 500 0

Solutions of the polymers from Examples 5, 6, and 7 were diluted withdeionized water to 1% solids by weight, pH 7. A 1% solids solution ofPEI (10,000 MW) in DI water, pH 7, was also prepared as a control.

A 5 mL polypropylene centrifuge tube was charged with 2.0 mL of BSAsolution, followed by 125 μL of diluted polymer solution. The centrifugetube was sealed and tumbled end over end for 30 minutes, thencentrifuged at 2000 rcf for 10 minutes. A BSA standard solution wasprepared by mixing 2 mL of original BSA solution with 125 μL of MOPSbuffer. A serial 1:1 dilution was performed to provide a total of 7 BSAstandards. These seven standards were pipetted (200 μL) in triplicateinto wells of a 96-well microtitration plate, along with triplicatesamples of the supernates from each polymeric flocculant beingevaluated. Three wells containing DI water as a blank were alsoincluded. The plate was analyzed using a SpectraMAX 250 MicroplateSpectrophotometer System (Molecular Devices Corp, Sunnyvale, Calif.)using a wavelength of 293 nm. Comparison of the absorptions of theflocculant solutions to those of the standards provided a measure of theflocculation efficiency. Results are recorded as the percentage ofstarting BSA remaining in solution; thus, the lower the number, thebetter the flocculant. Results are presented in the following Table 4:

TABLE 4 % BSA Remaining 0 mM 50 mM 100 mM 150 mM 200 mM 250 mM PolymerNaCl NaCl NaCl NaCl NaCl NaCl PEI (10,000) 0.0 40.6 87.2 96.1 101.6 99.7Example 5 0.9 6.2 29.3 53.9 83.4 96.8 Example 6 0.0 5.6 28.0 51.2 79.093.8 Example 7 0.3 18.2 40.3 60.8 78.1 88.3 Comparative 0.0 42.1 73.3101.9 103.1 99.3 Example 1This example illustrates that incorporation of as little as 2.5%guanidine groups into PEI dramatically improves its ability toprecipitate proteins in the presence of sodium chloride.

Example 34

Guanylated PEIs from Examples 11-13 were assayed for BSA precipitationby the procedure described in Example 33, except that 250 μL of 1%solids polymer solution was used instead of 125 μL. Results are shown inTable 4, compared to a control unmodified 70,000 MW PEI.

TABLE 5 % BSA Remaining 200 250 50 mM 100 mM 150 mM mM mM Polymer NaClNaCl NaCl NaCl NaCl PEI (70,000) 54.4 28.8 35.4 60.0 87.9 Example 1111.1 11.5 14.4 27.6 41.1 Example 12 17.7 7.4 9.0 13.1 95.6 Example 1317.6 16.7 20.2 27.8 39.1

Example 35

A Geobacillus stearothermophilus cell culture broth was provided by 3Mconsisting of approximately 1.4% by weight cell debris and spores. Testsamples of broth were prepared containing 0, 100, 200, and 300 mM NaClby a procedure similar to that described in Example 33. Solutions ofpolymers were prepared at 0.5% solids in DI water from 70,000 MW PEI andfrom the modified polymers of Examples 13, 16, and 20. A dilution seriesof each of these polymers was prepared (1:4, 1:4, 1:2, 1:2, 1:2) toprovide a total of 6 polymer concentrations. Then 2 mL of broth samplewas mixed with 0.5 mL of polymer solution, and the mixture was tumbledfor 30 minutes, then centrifuged at 200 rcf for 5 minutes. Standardswere prepared by mixing 2 mL of broth with 0.5 mL of DI water, carryingthe mixture through the same mixing/centrifugation procedure, thenpreparing a 2-fold serial dilution (6 samples) from the supernate.Supernates from the test solutions and from the standards were pipettedinto a 96-well microtitration plate and assayed by absorbancemeasurement at 650 nm. Comparison of the absorptions of the flocculantsolutions to those of the standards provided a measure of theflocculation efficiencies. Results are presented in the FIGS. 1-4 whichshow the removal of turbidity at different salt concentrations anddifferent polymer concentrations for an unmodified PEI polymer (FIG. 1),a guanylated PEI polymer (FIG. 2), an alkylated PEI polymer (FIG. 3),and a polymer that has been alkylated and guanylated (FIG. 4).

Similar results were observed for Examples 14, 15, 17, 18, 19, and 21;that is, alkylation of the PEI resulted in broadening the concentrationrange in which the flocculant was effective in higher saltconcentrations, while alkylation plus guanylation were synergistic inthis regard.

By making minor modifications to the assay procedure, similarflocculation studies on a Bacillus atrophaeus cell culture broth (2.2%by weight vegetative cells, cell debris, and spores), and a Clostridumsporogenes purified spore suspension (0.013% solids) were conducted andsimilar results were observed.

Example 36 Virus Flocculation

Aqueous suspensions of φX174 bacteriophage (ca. 10⁹ pfu/mL) wereprepared in 10 mM TRIS((hydroxymethyl)aminomethane) pH 8.0 containing 0,50 mM, and 150 mM NaCl. Aqueous solutions of flocculator polymers wereprepared in DI water, pH 7, at 0.001% polymer by weight. 16 μL ofpolymer solution were added to a 2 mL sample of bacteriophage suspensionin a centrifuge tube. The tube was sealed, vortexed, and rotatedend-over-end for 2 hours. The tubes were then centrifuged at 3000 rcffor 10 minutes, and the resultant suspensions were filtered through a0.45 micron sterile syringe filter (GHP Acrodisc, Pall Life Sciences). A10-fold dilution series was prepared.

One mL of each dilution was mixed with 1 mL E. coli culture (grown to anoptical density of 0.3-0.6 when measured at 550 nm). After waiting 5minutes, a sterile pipet was used to mix 4.5 mL TSA Top agar with thedilution/E. coli mixture and plated on TSB plates. After the top agarhad solidified, the plates were inverted and placed in a 37° C.incubator overnight. Plates were then removed from the incubator andφX174 plaques were counted and recorded. A dilution series of theoriginal virus suspension was also evaluated in a similar manner.Comparison of the results allowed estimation of the LRV (log reductionin viral load) as a result of the flocculant treatment. Results forseveral polymers are listed in Table 6:

TABLE 6 Virus LRV PhiX174 LRV 150 0 mM 50 mM mM Polymer NaCl NaCl NaClComparative 6.3 0.1 0.5 Example 1 Example 27 5.8 3.5 2.9 Example 11 8.53.4 2.0 Example 12 8.5 3.8 1.9 Example 13 >8 6.3 2.9

Example 37

Five Coating Baths were Prepared:

-   Coating Bath #1: 0.5% wt/wt    poly(2-acrylamido-2-methyl-1-propanesulfonic acid, sodium salt) in    deionized water;-   Coating Bath #2: 1% wt/wt polyethylenimine (10,000 M_(n)) in    deionized water;-   Coating Bath #'s 3-5: 1% wt/wt polymer of Examples 5, 6, or 7,    respectively, in deionized water.-   Aminosilane coated glass microscope slides (obtained from Newcomer    Supply, Middleton, Wis.) were dip coated, in sequence, with coating    solution #1, rinsed with deionized water, and dried at room    temperature. Slides were then dip coated in coating solution #2    (control), #3, #4, or #5, rinsed with deionized water, and dried at    room temperature. The coated slides could then be used to capture    bacteria or spores from aqueous media for enumeration or for    identification. A control aminosilane slide coated with only coating    solution #1 was found to bind almost no bacteria or spores.

Slides coated with PEI and the modified PEIs of Examples 5-7 wereevaluated for their ability to capture Clostridium sporogenes spores bythe following procedure:

-   -   Prepare a suspension of pure C. sporogenes spores at        approximately 1×10⁸ CFU/mL in 40% ethyl alcohol, 60% water.        Centrifuge between 0.5 and 1.0 mL of the spore suspension        (volume will depend on number of materials tested), discard the        supernatant and re-suspend 1:1 in 70% ethyl alcohol in water.        Next place three replicates of coated microscope slides into a        square Petri dish and apply 10 μl of the spore suspension 0.5 cm        from the bottom in the center of the slide. Leave for 1 minute        then rinse each slide under a steady stream of ultrapure water        from a Milli-Q system at a flow rate of approximately 1.2 liters        per minute for 10 seconds. Now dry the slide using a gentle flow        of nitrogen gas. Using a light microscope, place the        spore-exposed portion of the slide under the 10× objective and        randomly capture images of three separate areas within the        exposed region. Count the number of spores in each image;    -   this can be done by hand or alternately using image processing        software such as AxioVision (Zeiss). Calculate the average per        slide based on the counts from the three images on the same        slide. Finally, calculate the average and standard deviation per        material from the three replicates.        The results are displayed in Table 7:

TABLE 7 Coating: PEI Example 5 Example 6 Example 7 # of Spores 664 13921347 1050 Captured:

Example 38 p-Chlorophenylbiguanide Derivative of PEI

A solution of 2.00 grams of PEI (MW=10,000, from Polysciences, Inc.,Warrington, Pa.) in 10 mL of ethoxyethanol was treated with 2.93 mL of1.0 N aqueous hydrochloric acid solution.N³-p-chlorophenyl-N¹cyanoguanidine (570 mg, 2.94 mmol) was added and thereaction mixture was heated to 140° C. overnight. Thin layerchromatography indicated that all of theN³-p-chlorophenyl-N¹cyanoguanidine had been consumed. The reactionmixture was concentrated under reduced pressure to give an orange syrup.¹H-NMR indicated conversion to the p-chlorophenylbiguanide product. Theresulting syrup was dissolved in water to give a 20% by wt solutionbased on the PEI initial mass.

Evaluation of this polymer by the BSA precipitation test described inExample 33, using 250 μL flocculant solution, showed good BSA removal atall salt concentrations, up to 250 mM, tested.

Examples 39-44 Carbodiimide Modifications of PEI

A solution of 1.05 grams of PEI (MW=10,000, from Polysciences, Inc.,Warrington, Pa.) in 10 mL of tert-butyl alcohol was placed in a vial andtreated with enough dicyclohexycarbodiimide (314 mg, 1.52 mmol) to reactwith 6.3% of the amine groups. The vial was sealed and the mixture washeated at 100° C. overnight. The reaction mixture was concentrated underreduced pressure to give a colorless syrup. ¹H-NMR indicated conversionto the dicyclohexyl guanide product (Example 39). Likewise, PEI samplesfunctionalized with 12.5% and 25% dicyclohexyl guanides were alsoprepared (Examples 40 and 41, respectively). The resulting syrups with6.3 and 12.5% dicyclohexyl guanides were dissolved in dilutehydrochloric acid to give a 10% by wt solution based on the initial PEImass. The product with 25% dicyclohexyl guanides was dissolved in 1:1ethanol/dilute hydrochloric acid to give a 5% by wt solution based onthe initial PEI mass.

A solution of 0.99 grams of PEI (MW=10,000, from Polysciences, Inc.,Warrington, Pa.) in 10 mL of tert-butyl alcohol was placed in a vial andtreated with enough N-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (280 mg, 1.46 mmol) to react with 6.3% of the aminegroups. The vial was sealed and the mixture was heated at 100° C.overnight. The reaction mixture was concentrated under reduced pressureto give a colorless syrup. ¹H-NMR indicated conversion to the desiredguanide product (Example 42). Likewise, PEI samples functionalized with12.5% and 25% N-[3-(dimethylamino)propyl]-3-ethylcarbodiimide were alsoprepared (Examples 43 and 44, respectively). The resulting syrups weredissolved water to give a 10% by wt solution based on the initial PEImass.

Polymers of Examples 39 and 41 were diluted to 1% solids and evaluatedfor BSA precipitation using 250 μL flocculant solution. Results aredisplayed in Table 8, along with those for unmodified PEI:

TABLE 8 % BSA Remaining 50 mM 100 mM 150 mM 200 mM 250 mM Polymer NaClNaCl NaCl NaCl NaCl PEI (10,000) 4.0 12.5 40.0 83.7 92.8 Example 39 3.810.5 27.3 63.6 80.9 Example 41 4.6 8.0 17.6 39.9 47.0

Example 45

The polymer of Example 11 was diluted to 0.5% solids with isopropanol.Four portions of this solution (50 grams each) were formulated withenough butanediol diglycidyl ether (BUDGE) to react with 2.5%, 5%, 10%,and 20%, respectively, of the amine groups of the polymer. Samples (ca.10 cm×10 cm) of a nylon 66 membrane (single reinforced layer nylon threezone membrane, nominal pore size 1.8 μm, from 3M Purification Inc,Meridan, Conn.), were dip coated with the polymer solution, excesscoating solution was removed using a #14 wire-wound coating rod, thenallowed to dry for 15 minutes. In some instances, a second coating layerwas applied. The coated membranes were then placed in 500 mLpolyethylene bottles filled with deionized water and allowed to mixovernight to extract any non-crosslinked coating. Disks (24 mm diameter)were punched out of the membranes and placed in 5 mL centrifuge tubes.Bovine serum albumin solution (BSA, Sigma Aldrich) was prepared to aconcentration of 1.1 mg/ml in 25 mM TRIS buffer, pH 8.0(tris(hydroxymethyl)aminomethane, Sigma). 4.5 ml of the BSA solution waspipetted into each centrifuge tube, the tubes were capped, and the tubeswere tumbled overnight. The supernatant solutions were analyzed by aUV-VIS spectrometer at 279 nm with background correction applied at 325nm. Static binding capacities for the samples are listed in Table alongwith that for an uncoated membrane.

TABLE 9 % Crosslinker # of Coating Static BSA Capacity (BUDGE) Layers(mg/mL) 2.5 1 15 2.5 2 20 5 1 17 5 2 26 10 1 19 10 2 35 20 1 27 20 2 460 0 1

Examples 46-48 Cyanoguanidine-Derivatized PEI

A solution of 2.01 grams of PEI (MW=10,000, from Polysciences, Inc.,Warrington, Pa.) in 11.7 mL of 0.1 N aqueous hydrochloric acid wasplaced in a pressure flask and treated with enough sodium dicyanamide(104 mg, 1.17 mmol) to react with 2.5% of the amine groups. The flaskwas sealed and the mixture was heated at 120° C. for 5 hours. ¹H-NMRindicated conversion to the cyanoguanidine product (Example 46).

Likewise, a 1.96 g solution of PEI dissolved in 9 mL of water wastreated with 2.9 mL of 1.0 N hydrochloric acid and sodium dicyanamide(255 mg, 2.86 mmol) to give a product where 6.3% of the amines wereconverted to cyanoguanides and a 1.99 g solution of PEI dissolved in 6mL of water was treated with 5.8 mL of 1.0 N hydrochloric acid andsodium dicyanamide (520 mg, 5.84 mmol) to give a product where 12.5% ofthe amines were converted to cyanoguanides (Examples 47 and 48,respectively).

Examples 49-51 Urea Modification of PEI

A solution of 3.00 grams of PEI (MW=10,000, from Polysciences, Inc.,Warrington, Pa.) in 15 mL of CH₂Cl₂ was treated with enoughtrimethylsilyl isocyante (235 μL, 1.74 mmol) to react with 2.5% of theamine groups. After stirring for 1 h, the reaction mixture was treatedwith a few drops of methanol and concentrated under reduced pressure.¹H-NMR indicated conversion to the urea product (Example 49). Theresulting syrup was dissolved in water to give a 20% by wt solutionbased on the PEI initial mass. Likewise, PEI functionalized with 6.3%and 12.5% ureas were also prepared (Examples 50 and 51, respectively).

Examples 52-54

The urea modified PEI materials from Examples 49-51, were each reactedwith enough pyrazole-1-carboxamidine hydrochloride, by proceduressimilar to that used in Example 1, to convert 5% of the amine groups toguanidines. ¹H-NMR indicated conversion to the expected derivatizedproducts (Examples 52-54, respectively).

Example 55 PEI Biguanide

A solution of 92 mg of PEI (MW=10,000, from Polysciences, Inc.,Warrington, Pa.) in 0.9 mL of water was placed in a vial and treatedwith N-amidinopyrazole-carboxamidine hydrochloride (100 mg, 0.53 mmol)to react with 25% of the amine groups. The vial was sealed and themixture was heated at 100° C. overnight. ¹H-NMR indicated conversion tothe desired guanide product.

Example 56 Poly(polyethylenimine Biguanide)

A solution of 1.00 grams of PEI (MW=600, from Polysciences, Inc.,Warrington, Pa.) in 10 mL of water was placed in a pressure flask andtreated with sodium dicyanamide (139 mg, 1.56 mmol) and 200 μL of aceticacid. The flask was sealed and the mixture was heated at 140° C.overnight. ¹H-NMR indicated conversion to the polybiguanidine product.

In a similar manner, PEI polymers of different molecular weights can beutilized, and differing ratios of PEI to sodium dicyanamide can beutilized, to prepare a variety of poly(PEI biguanide)s.

Examples 57-59 Modification with Guanidinoacetic Acid

Guanidinoacetic acid (6.0 grams) was dissolved in 1 N aqueoushydrochloric acid (51.4 mL).2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (14 grams) was dissolvedin an ethanol (25 grams)/methanol (11 grams) mixture. The two solutionswere then mixed, and allowed to react for 10 minutes. A portion of thismixture (15.3 grams) was added to a PEI solution (16.67 grams of a 30%solids 70,000 MW PEI solution in water). This mixture was allowed toreact for 6 hours to acylate 6.3% of the amine groups of the PEI(Example 57). By similar procedures, modified polymers having 12.5% and20% of the amine groups acylated were prepared (Examples 58 and 59,respectively).

A portion of the polymer solution of Example 59 was diluted to 1% byweight in deionized water, pH 7, and evaluated in the BSA precipitationtest, providing excellent flocculation at all salt concentrations.

Example 60

Using standard microbiological procedures, cultures of the followingwere prepared:

a) Escherichia coli (cells and cell debris)

b) Chinese hamster ovary (CHO) cells

c) Baker's yeast

When flocculation experiments were conducted similarly to thosedescribed in Example 35 on these mixtures, the ligand functionalpolymers of the invention consistently displayed good flocculatingability in the presence of sodium chloride concentrations in excess of50 mM.

The invention claimed is:
 1. A method of separating a target biologicalspecies from a fluid comprising: contacting the fluid with a substratehaving a coating on a surface thereof of a ligand-functionalized polymercomprising a water soluble or water dispersible aminopolymerfunctionalized with guanidinyl groups; whereby a complex comprising theligand-functionalized polymer and the target biological species isformed, and separating the complex; wherein said target biologicalspecies selected from biomacromolecules and microbiological species,wherein the substrate is selected from a film, or a woven or nonwovenweb.
 2. The method of claim 1 wherein said biomacromolecules areselected from proteins, enzymes, nucleic acids, and endotoxins.
 3. Themethod of claim 1 wherein said microbiological species is selected frombacteria, viruses, cells, cell debris, and spores.
 4. The method ofclaim 1 wherein the aminopolymer functionalized with guanidinyl groupsis of the formula:

wherein R² is a H, C₁-C₁₂ alkyl, C₅-C₁₂ (hetero)aryl, or a residue ofthe polymer chain; each R³ is independently H, C₁-C₁₂ alkyl, or C₅-C₁₂(hetero)aryl, each R⁴ is H, C₁-C₁₂ alkyl or alkylene, C₅-C₁₂(hetero)aryl or (hetero)arylene, cyano, or —C(=NH)—N(R²)-Polymer, and nis 1 or
 2. 5. The method of claim 1 wherein the aminopolymer is selectedfrom the group consisting of polyethylenimine, polylysine,polyaminoamides, polyallylamine, polyvinylamine,polydimethylamine-epichlorohydrin-ethylenediamine, polyaminosiloxanesand dendrimers formed from polyamidoamine (PAMAM) and polypropylenimine.6. The method of claim 1 wherein the base substrate is porous.
 7. Themethod of claim 6 wherein the porous base substrate is a microporousbase substrate.
 8. The method of claim 6 wherein the porous basesubstrate is a nonwoven web.
 9. The method of claim 3 wherein the cellsare selected from archaea, bacteria, and eucaryota.
 10. The method ofclaim 1 wherein the fluid is derived from a cell culture or fermentationprocess.
 11. The method of claim 1 wherein the fluid comprises asolution of a purified protein or enzyme after separating the complex.12. The method of claim 1 wherein the separated complex comprises apurified protein or enzyme.
 13. The method of claim 10 wherein the fluidhas a salt content of at least 50 millimolar.
 14. The method of claim 10wherein the fluid has a salt content of at least 100 millimolar.
 15. Themethod of claim 1 wherein the amount of ligand-functionalized polymerrelative to the amount of target biological species is 0.01% to 100% byweight.
 16. The method of claim 1 wherein the ligand-functionalizedpolymer is present in amounts of 0.01 to 5000 micrograms/mL of fluid.17. The method of claim 1 wherein 0.1 to 100 mole percent of theavailable amino groups of the aminopolymer is functionalized withguanidinyl groups.
 18. The method of claim 1 wherein functionalizedguanidinyl groups are pendent from the polymer chain.
 19. The method ofclaim 1 wherein functionalized guanidinyl groups are in the aminopolymerchain.
 20. The method of claim 1 wherein the ligand functionalizedpolymer is prepared by the steps of contacting a polyamine polymer witha guanylating agent selected from cyanamide; O-alkylisourea salts;chloroformamidine hydrochloride; 1-amidino-1,2,4-triazole hydrochloride;3,5-dimethylpyrazole-1-carboxamidine nitrate; and carbodiimides.
 21. Themethod of claim 20 further comprising the step of alkylating oracylating a portion of the amino groups of the ligand-functionalizedpolymer.